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          基因工程大纲
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        <h1 id="基因工程"><a href="#基因工程" class="headerlink" title="基因工程"></a>基因工程</h1><h2 id="前言"><a href="#前言" class="headerlink" title="前言"></a>前言</h2><h3 id="技术"><a href="#技术" class="headerlink" title="技术"></a>技术</h3><ul>
<li>DNA重组</li>
<li>转基因</li>
</ul>
<h3 id="操作水平"><a href="#操作水平" class="headerlink" title="操作水平"></a>操作水平</h3><ul>
<li>DNA分子水平</li>
</ul>
<h2 id="DNA重组技术的基本工具"><a href="#DNA重组技术的基本工具" class="headerlink" title="DNA重组技术的基本工具"></a>DNA重组技术的基本工具</h2><h3 id="限制性核酸内切酶"><a href="#限制性核酸内切酶" class="headerlink" title="限制性核酸内切酶"></a>限制性核酸内切酶</h3><h3 id="DNA连接酶"><a href="#DNA连接酶" class="headerlink" title="DNA连接酶"></a>DNA连接酶</h3><ul>
<li>E·coil DNA连接酶</li>
<li>T_4 DNA连接酶</li>
</ul>
<h3 id="基因进入受体细胞的载体"><a href="#基因进入受体细胞的载体" class="headerlink" title="基因进入受体细胞的载体"></a>基因进入受体细胞的载体</h3><h2 id="基因工程的基本操作程序"><a href="#基因工程的基本操作程序" class="headerlink" title="基因工程的基本操作程序"></a>基因工程的基本操作程序</h2><h3 id="目的基因的获取"><a href="#目的基因的获取" class="headerlink" title="目的基因的获取"></a>目的基因的获取</h3><ul>
<li>从自然界已有的物种中分离出来</li>
<li>人工的方法合成</li>
<li><p>常用方法</p>
<ul>
<li><p>从基因文库中获取目的基因</p>
<ul>
<li>基因组文库</li>
<li>部分基因文库</li>
</ul>
</li>
<li><p>利用PCR技术扩增目的基因</p>
<ul>
<li>全称：多聚酶链式反应</li>
<li>前提：要有一段已知目的基因的核苷酸序列-&gt;合成引物</li>
<li>过程：变性-&gt;退火-&gt;延伸</li>
<li>原料：模板、引物、Taq酶、脱氧核苷三磷酸（脱氧核苷酸）、缓冲液</li>
<li>PS：引物不可过长/过短，一对引物不可互补</li>
<li>PPS：Taq酶来源于水生嗜热杆菌Taq</li>
</ul>
</li>
<li><p>DNA合成仪用化学方法直接人工合成</p>
<ul>
<li>只适用于短的</li>
<li>不需要模板</li>
</ul>
</li>
</ul>
</li>
</ul>
<h3 id="基因表达载体的构建（核心）"><a href="#基因表达载体的构建（核心）" class="headerlink" title="基因表达载体的构建（核心）"></a>基因表达载体的构建（核心）</h3><ul>
<li>目的：使目的基因在受体细胞中稳定存在；遗传；使目的基因表达和发挥作用</li>
<li><p>基因表达载体</p>
<ul>
<li>目的基因</li>
<li>启动子、终止子</li>
<li>标记基因</li>
<li>复制原点</li>
</ul>
</li>
</ul>
<h3 id="将目的基因导入受体细胞"><a href="#将目的基因导入受体细胞" class="headerlink" title="将目的基因导入受体细胞"></a>将目的基因导入受体细胞</h3><ul>
<li>目的基因导入受体细胞内，并且受体细胞内维持稳定和表达的过程，称为转化</li>
<li><p>导入植物细胞</p>
<ul>
<li><p>农杆菌转化法</p>
<ul>
<li>适用于双子叶和裸子植物（受伤时分泌酚类化合物吸引农杆菌）</li>
<li>农杆菌上的Ti质粒上的T-DNA（可转移的DNA）可转移至受体细胞并整合到染色体DNA上</li>
</ul>
</li>
<li><p>基因枪法</p>
<ul>
<li>常用于单子叶植物</li>
</ul>
</li>
<li><p>花粉管通道法</p>
<ul>
<li>我国转基因抗虫棉就是这么获得的</li>
<li>十分简便经济</li>
</ul>
</li>
</ul>
</li>
<li><p>导入动物细胞</p>
<ul>
<li><p>显微注射技术</p>
<ul>
<li>先给受精卵注射，经胚胎早期培养一段时间后再移植到雌性动物的输卵管或子宫内</li>
</ul>
</li>
</ul>
</li>
<li><p>导入微生物细胞</p>
<ul>
<li><p>感受态细胞法</p>
<ul>
<li>原核生物特点：繁殖快、多为单细胞、遗传物质相对较少</li>
<li><p>过程</p>
<ul>
<li>先用钙离子增大细菌细胞壁的通透性</li>
<li>再将重组表达载体DNA分子溶于缓冲液中与感受态细胞混合，在一定温度下促进感受态细胞吸收DNA分子</li>
</ul>
</li>
</ul>
</li>
</ul>
</li>
</ul>
<h3 id="目的基因的检测与鉴定"><a href="#目的基因的检测与鉴定" class="headerlink" title="目的基因的检测与鉴定"></a>目的基因的检测与鉴定</h3><ul>
<li><p>分子检测</p>
<ul>
<li>DNA上是否插入了目的基因（DNA分子杂交技术）</li>
<li>目的基因是否转录出了mRNA（同样是分子杂交技术）</li>
<li>目的基因是否翻译成蛋白质（抗原-抗体杂交）</li>
</ul>
</li>
<li><p>个体生物学水平的鉴定</p>
</li>
</ul>
<h2 id="基因工程的应用"><a href="#基因工程的应用" class="headerlink" title="基因工程的应用"></a>基因工程的应用</h2><h3 id="植物基因工程硕果累累"><a href="#植物基因工程硕果累累" class="headerlink" title="植物基因工程硕果累累"></a>植物基因工程硕果累累</h3><ul>
<li><p>抗虫转基因植物</p>
<ul>
<li><p>Bt 毒蛋白基因</p>
<ul>
<li>分离于苏云金芽孢杆菌</li>
<li>在昆虫肠道的碱性环境中才表现出毒性</li>
</ul>
</li>
<li><p>蛋白酶抑制剂基因</p>
<ul>
<li>使昆虫不消化蛋白质</li>
</ul>
</li>
<li><p>淀粉酶抑制剂基因</p>
<ul>
<li>使昆虫不消化淀粉，阻断能量来源</li>
</ul>
</li>
<li><p>植物凝集素基因</p>
</li>
</ul>
</li>
<li><p>抗病转基因植物</p>
<ul>
<li><p>抗病毒</p>
<ul>
<li><p>病毒外壳蛋白</p>
<ul>
<li>把病毒包起来</li>
</ul>
</li>
<li><p>病毒的复制酶基因</p>
</li>
</ul>
</li>
<li><p>抗真菌</p>
<ul>
<li>几丁质酶基因</li>
<li>抗毒素合成基因</li>
</ul>
</li>
</ul>
</li>
<li><p>抗逆转基因植物</p>
<ul>
<li>调节细胞渗透压的基因</li>
<li>鱼的抗冻蛋白基因</li>
<li>抗除草剂基因</li>
</ul>
</li>
<li><p>改良植物的品质</p>
<ul>
<li><p>提高氨基酸含量</p>
<ul>
<li>将必需氨基酸含量多的蛋白质编码基因导入植物</li>
<li>改变这些氨基酸合成途径中某种关键酶的活性</li>
</ul>
</li>
<li><p>转基因延熟番茄</p>
<ul>
<li>控制果实成熟的基因</li>
</ul>
</li>
<li><p>转基因矮牵牛颜色变异</p>
<ul>
<li>花青素代谢有关的基因</li>
</ul>
</li>
</ul>
</li>
</ul>
<h3 id="动物基因工程前景广阔"><a href="#动物基因工程前景广阔" class="headerlink" title="动物基因工程前景广阔"></a>动物基因工程前景广阔</h3><ul>
<li><p>用于提高动物生长速度</p>
<ul>
<li>外源生长激素基因</li>
</ul>
</li>
<li><p>用于改善畜产品的品质</p>
<ul>
<li>肠乳糖酶基因</li>
</ul>
</li>
<li><p>用于转基因动物生产药物</p>
<ul>
<li><p>乳腺生物反应器（乳房生物反应器）PS：只在泌乳期生效</p>
<ul>
<li>将药用蛋白基因与乳腺蛋白基因的启动子等调控组件连接在一起</li>
</ul>
</li>
</ul>
</li>
<li><p>用转基因动物作器官移植的供体</p>
<ul>
<li>难题：克服免疫排斥</li>
<li><p>方法</p>
<ul>
<li>1.将器官供体基因组导入某种调节因子，以抑制抗原决定基因的表达</li>
<li>2.设法除去抗原决定基因，再结合克隆技术，培育出没有免疫排斥反应的转基因克隆猪器官</li>
</ul>
</li>
</ul>
</li>
</ul>
<h3 id="基因工程药物异军突起"><a href="#基因工程药物异军突起" class="headerlink" title="基因工程药物异军突起"></a>基因工程药物异军突起</h3><ul>
<li>工程菌：用基因工程的方法，使外源基因得到高效率表达的菌类细胞株系</li>
<li>干扰素：一种糖蛋白，几乎能抵抗所有病毒引起的感染</li>
<li>PS：DNA重组技术生产的一般是人类蛋白药物</li>
</ul>
<h3 id="基因治疗曙光初照"><a href="#基因治疗曙光初照" class="headerlink" title="基因治疗曙光初照"></a>基因治疗曙光初照</h3><ul>
<li>方法：把正常基因导入病人体内，使该基因的表达产物发挥功能，从而治疗疾病，是治疗遗传病的最有效的手段</li>
<li><p>基因治疗类型</p>
<ul>
<li><p>体外基因治疗</p>
<ul>
<li><p>过程</p>
<ul>
<li>1.从病人体内获得某种细胞，例如T淋巴细胞，进行培养</li>
<li>2.在体外完成基因转移</li>
<li>3.筛选成功转移的细胞扩增培养</li>
<li>4.重新输入患者体内</li>
</ul>
</li>
<li><p>操作复杂但效果较为可靠</p>
</li>
</ul>
</li>
<li><p>体外基因治疗</p>
<ul>
<li>直接向人体组织细胞中转移基因</li>
</ul>
</li>
</ul>
</li>
<li><p>用于基因治疗的基因种类</p>
<ul>
<li><p>从健康人体上分离的得到的功能正常的基因</p>
<ul>
<li>弥补缺陷</li>
</ul>
</li>
<li><p>反义基因</p>
<ul>
<li>与病变mRNA互补，阻断非正常蛋白质合成</li>
</ul>
</li>
<li><p>自杀基因</p>
<ul>
<li>编码可以杀死癌变细胞的蛋白酶基因</li>
</ul>
</li>
</ul>
</li>
</ul>
<h2 id="蛋白质工程的崛起"><a href="#蛋白质工程的崛起" class="headerlink" title="蛋白质工程的崛起"></a>蛋白质工程的崛起</h2><h3 id="概念"><a href="#概念" class="headerlink" title="概念"></a>概念</h3><ul>
<li>指以蛋白质分子的结构规律及其与生物功能的关系作为基础，通过基因修饰或基因合成，对现有蛋白质进行改造，或制造一种新的蛋白质，以满足人类的生产和生活的需求</li>
</ul>
<h3 id="崛起的缘由"><a href="#崛起的缘由" class="headerlink" title="崛起的缘由"></a>崛起的缘由</h3><ul>
<li>基因工程原则上只能生产自然界已存在的蛋白质</li>
</ul>
<h3 id="基本原理"><a href="#基本原理" class="headerlink" title="基本原理"></a>基本原理</h3><ul>
<li>目标：根据人们对蛋白质功能的特定需求，对蛋白质结构进行分子设计</li>
<li>因为基因决定蛋白质，最终还必须通过基因来完成</li>
<li><p>基本途径</p>
<ul>
<li>1.从预期的蛋白质功能出发</li>
<li>2.设计预期的蛋白质结构</li>
<li>3.推测应有的氨基酸序列</li>
<li>4.找到相对应的脱氧核苷酸序列（基因）</li>
</ul>
</li>
</ul>
<h3 id="目前局限"><a href="#目前局限" class="headerlink" title="目前局限"></a>目前局限</h3><ul>
<li>目前科学家对大多数蛋白质的高级结构的了解还很不够</li>
</ul>

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